Antifolates, a specific class of antimetabolite, have had an important impact on the treatment of cancer. They act as chemotherapeutic agents by interference with normal folate metabolism which in turn prevents tumor cell function and replication. One specific effect of these drugs on folate metabolism is denial of methylenetetrahydrofolate, a cofactor form which is necessary for the thymidylate synthase mediated production of thymidylate and hence DNA synthesis. However, a complete understanding of their mechanism of action will require quantitative measurement of essentially all intracellular folates, and their polyglutamate conjugates, in eucaryotic cell systems that can be exposed to antifolates under rigorously defined conditions. During previous work on this project we have developed a unique method to measure these particularly unstable tissue reduced folates based upon entrapment of cell free-extract methylenetetrahydrofolate into a covalent complex with thymidylate synthase that has been radiolabeled with tritiated fluorodeoxyuridylate. This highly sensitive method permits not only quantitative determination of reduced folates but evaluation of their polyglutamates as well by electrophoretic separation of labeled complexes. We intend to apply these methods to an investigation of the impact of methotrexate and other antifolates on the level and polyglutamate status of five specific reduced folate pools in hepatoma cells grown in culture. The folate pools which will be cycled to the methylene form for subsequent analysis are dihydrofolate, tetrahydrofolate, methylenetetrahydrofolate, methyltetrahydrofolate and formyltetrahydrofolate. These folates and their polyglutamates will be manipulated intracellularly through changes in media folates in conjunction with drug exposure to provide maximal opportunity to observe the impact of antifolates on specific folate pools. It is proposed that such a study will provide a far more accurate and quantitative understanding of the mode of action of antifolates and also valuable information about how to determine dose level and temporal optimization of drug usage in the treatment of cancer.